US5681559A - Method for producing a highly enriched population of hematopoietic stem cells - Google Patents
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Definitions
- This invention is related to the isolation of a cell population enriched in hematopoietic stem cells.
- Mammalian hematopoietic cells are responsible for an extraordinarily diverse range of activities. They are divided into several lineages, including lymphoid, myeloid and erythroid.
- the lymphoid lineage comprising B cells and T cells, produces antibodies, regulates cellular immunity, and detects foreign agents such as disease-causing organisms in the blood.
- the myeloid lineage which includes monocytes, granulocytes, and megakaryocytes, monitors the blood for foreign bodies, protects against neoplastic cells, scavenges foreign materials, and produces platelets.
- the erythroid lineage includes red blood cells, which carry oxygen.
- stem cell a single cell type called the hematopoietic "stem cell” is believed to act as the progenitor of all hematopoietic lineages.
- These rare primitive cells approximately 0.01% of bone marrow cells
- Stem cells differentiate into multipotent progenitor cells and ultimately into each of the mature hematopoietic lineages.
- stem cells are believed to be capable of generating long-term hematopoiesis when transplanted into immunocompromised hosts.
- the stem cell was originally defined by the capacity to self-renew and to give rise to progeny that are the committed precursors for all hematopoietic lineages.
- a number of researchers have concluded from their attempts to divide the progenitor cell compartment into stem cell and committed progenitor cells that these compartments constitute a hierarchy or continuum of cell types whose maturation is characterized by decreasing pluripotentiality and by a decreasing ability to repopulate the hematopoietic system of serially transplanted animals.
- stem cells typically seek to exploit differences in cell size or density or the selection or depletion of cells based on the expression of cell surface antigens. It has been difficult, however, to identify and purify stem cells because of the small proportion of stem cells in the bone marrow, peripheral blood, and other sources. In addition, many cell surface markers associated with stem cells are also present on more differentiated cells.
- CD34 for example, is thought to be present on all human hematopoietic progenitor cells (Civin et al. (1984) J. Immunol. 133:157-165), and this population can mediate engraftment of an immunocompromised host in vivo (Berenson et al. (1991) Blood 77:1717-1722). Although the presence of primitive hematopoietic cells expressing relatively high CD34 density has been reported (Berenson et al. (1991); Terstappen et al. (1991) Blood 77:1218-1227; Teixido et al. (1992) J. Clin. Invest.
- the CD34 + cell population is heterogeneous with respect to the types of progenitor cells and their relative state of differentiation (Terstappen et al. (1991)) and the fraction of the CD34 + compartment containing hematopoietic stem cells has not been consistently and reliably defined.
- hematopoietic stem cells The relative paucity of hematopoietic stem cells has prevented extensive research on stem cells and hematopoietic differentiation in general.
- the ready availability of a cell population enriched in hematopoietic stem cells would make possible the identification of biological modifiers affecting stem cell behavior. For example, there may be as yet undiscovered growth factors associated with (1) early steps of dedication of the stem cell to a particular lineage; (2) the prevention of such dedication; and (3) the ability to control stem cell proliferation.
- stem cells are also important targets for gene therapy.
- the present invention provides methods for obtaining cell populations, preferably human cells, especially fetal cells, enriched in hematopoietic stem cells selected on the basis of possession of mean fluorescence values (MFV) for CD34 surface antigen approximately 100 times or more than that of isotype controls.
- MMV mean fluorescence values
- compositions obtained by such methods are useful, for example, in reconstituting hematopoiesis in an animal lacking a functioning hematopoietic system. These compositions are also useful for treating an animal affected by a genetic disease comprising introducing into the animal a CD34 hi cell transfected with a nucleic acid capable of either expressing in the transfected cell a polypeptide which is missing or defective in the animal or expressing a nucleic acid or polypeptide capable of inhibiting the expression of a target protein in the animal.
- Also provided are methods for evaluating a sample for the presence of a biological modifier capable of affecting a biological response of a hematopoietic stem cell the method comprising plating a test CD34 hi cell (with the sample) and a control CD34 hi cell (without the sample) in an appropriate culture system and comparing the biological response of the test and control CD34 hi cells.
- FIG. 1 shows the distribution of CD34 and lineage markers as determined by flow cytometry on an (A) isotype control and (B) on low density fetal bone marrow cells stained with anti-CD34 antibody (Tuk-3) and lineage antibodies (CD14, CD15, CD16).
- FIG. 1(B) shows that there are two populations of CD34 + cells, those expressing high levels of CD34 (arrow) and those expressing relatively low levels of CD34 (box).
- FIG. 2 shows a histogram of the CD34 fluorescence distribution of CD34 hi and CD34 lo sorted by flow cytometry. Sorted CD34 lo and CD34 hi cells were reanalyzed and the percentage overlap calculated using Multiplus Software according to the Overton subtraction procedure. The shaded portion of the curves indicates the non-overlapping portion of the two populations and hence the relative purity of the samples.
- FIG. 3 shows phenotypic analysis of CD34 hi and CD34 lo cells.
- Cells sorted by flow cytometry were collected and restained with directly conjugated antibodies to other cell surface markers: CD2, CD10, CD19, CD13, CD33, CD38, HLA-DR, CD45RA, and Thy-1.
- FIG. 4 lists the precursor frequencies for sorted fetal bone marrow subpopulations as the percentage of cells that form cobblestone areas between weeks four and six in SyS-1 coculture as determined by limiting dilution analysis. Some cultures were either lost to contamination or sacrificed for analysis prior to week 6 (blank spaces). The average response (AVG) and standard deviation (SD) were calculated. The number of cells containing one cobblestone area-forming cell is listed as the reciprocal of the average frequency (1/AVG).
- FIG. 5 shows phenotypic analysis of bulk cultures of CD34 hi cells.
- CD34 hi cells were cultured on a stromal cell layer for six weeks, then the entire culture was harvested and stained with either the appropriate isotype matched antibodies (A), anti-CD10 and anti-CD19 (B), or anti-CD15 and anti-CD33 (C). Stromal cells and dead cells were excluded by light scatter and dye exclusion gating.
- FIG. 6 shows FACS analyses of cells from a representative SCID-hu bone graft injected with 5 ⁇ 10 4 allogeneic CD34 hi fetal bone marrow cells and retrieved after nine weeks.
- Cells were stained with CD45 and W6-32 or donor HLA (HLA-D) and W6-32 antibodies, (top left panels). Gating on the donor population revealed a heterogeneous distribution of bone marrow cells (upper right panel). B-cells were identifiable by expression of CD19 and CD20. Gating on CD20 + donor cells showed forward (FSC) and side scatter (SSC) characteristics of B-cells (middle right panel).
- CD33 + donor cells were observed in the expected myeloid region of bone marrow cells (bottom left panel).
- CD14 + monocytes had characteristic size and granularity features (lower middle right panel).
- a population of CD34 + donor cells was identifiable (14% of total sample); the specificity of staining was controlled by lack of immunoreactivity with irrelevant antibodies (bottom right panel).
- FIG. 7 shows engraftment of CD34 subsets in the SCID-Hu thymus assay.
- Depleted thymic fragments were injected with 1 ⁇ 10 4 fetal bone marrow CD34 hi or CD34 lo cells and implanted into SCID mice.
- Thymic grafts were retrieved after 6 to 14.5 weeks and analyzed for the combined expression of CD45, W6-32 and HLA-D. These cells also express T-cell specific markers, either CD1, CD3, CD4, or CD8.
- FIG. 8 shows FACS analyses of two representative SCID-Hu thymus grafts, one injected with CD34 hi cells (right panels, top to bottom) and the other unsuccessfully reconstituted with CD34 lo cells (left panels--top to bottom).
- the present invention provides a method for isolating a population of hematopoietic cells highly enriched for stem cells by separating two distinct populations of CD34 + cells, one expressing high levels of CD34 antigen ("CD34 hi ”) and the other expressing lower levels of CD34 antigen ("CD34 lo ").
- CD34 + cells are being fractionated based on calculations related to relative CD34 antigen density and not by the mere percentage of CD34 + cells in a cell population or another arbitrary cutoff.
- CD34 hi cells are a discrete population of cells forming only about 2% of low density fetal bone marrow mononuclear cells.
- a simple single step fractionation of fetal bone marrow based on levels of CD34 expression provides a high yield of cells highly enriched in stem cell activity.
- CD34 + cells When CD34 + cells are separated into the “high” and “low” fractions, the stem cells which serve as the progenitors for all human hematopoietic cell lineages are found exclusively in the CD34 hi fraction. Long-term multilineage potential is exclusively contained in the CD34 hi compartment. As shown in the examples below, a much higher percentage of CD34 hi cells score positive in long term in vitro stromal coculture assays than for CD34 + or CD34 lo cells. Additionally, a higher percentage of CD34 hi cells exclusively engraft into allogeneic fetal bone fragments implanted into severe combined immunodeficiency (SCID) mice and provide long term myelopoiesis and B-lymphopoiesis. Finally, more CD34 hi cells differentiate into T cells in allogeneic thymus grafts implanted into SCID mice.
- SCID severe combined immunodeficiency
- CD34 lo cells do not display any significant long term activity in the generation of B cells or myeloid cells in vitro. Moreover, CD34 lo cells are incapable of maintaining long term hematopoiesis in human bones and do not possess T cell progenitor activity. In short, all stem cell activity appears to be confined to the CD34 hi cell population.
- CD34 hi cells were highly enriched for the phenotypes that have been reported to define the most primitive hematopoietic cells, such as CD34 + /Thy-1 + (Baum et al., 1992), CD34 + /HLA-DR lo (Brandt et al. (1988); CD34 + /CD38 lo (Terstappen et al., 1991), CD34 + /CD45 RA - (Landsdorp and Dragowska (1992). While these cells also express low levels of CD13 and CD33, which are found on multilineage progenitors, they do not bear cell surface antigens that define mature cells (CD2, CD10, CD14, CD15, CD16, CD19, or CD20).
- CD34 lo cells express antigens that suggest that they are activated progenitors for B and myeloid cells (CD10, CD19, and high levels of HLA-DR and CD38). CD34 lo cells also do not express CD2; in that respect our data differ from what was reported by Terstappen et al. (1992), since we do not seem to identify a CD34 + /CD2 + population in fetal bone marrow.
- the differential expression of lineage antigens on CD34 subsets underscores the fact that CD34 hi cells form a biologically distinct population.
- a CD34 hi cell preparation is one which contains approximately 90% or more, and preferably 95% or more, CD34 hi cells.
- CD34 hi cells are preferably prepared from fetal hematopoietic cell sources, e.g., bone marrow or liver, but may be purified from other fetal, neonatal, or adult hematopoietic cell sources, including bone marrow, fetal liver, embryonic yolk sac, fetal and adult spleen, and blood.
- Bone marrow cells may be obtained from the tibia, femur, spine, or other bone cavities.
- CD34 antigen in adult tissue is variable, depending on the differentiation state of any given cell in the sample.
- CD34 negative cells hose expressing levels of CD34 antigen indistinguishable from the background
- CD34 + cells express a range of antigen density much like that observed in fetal bone marrow, with the exception that the maximum CD34 antigen density for adult hematopoietic cells is somewhat less than that seen in fetal tissue, generally less than 100-fold higher than isotype controls.
- the adult cells having the highest cell surface density of CD34 do not form a clearly demarcated cell population on that basis alone, and thus a second marker is required to better define the population of cells possessing all hematopoietic stem cell activity.
- CD34 hi cells from fetal tissues have low levels of CD33 and CD38; intermediate levels of HLA-DR and CD13; and no appreciable CD14, 15, 16, glycophorin A.
- Stem cells are Lin - .
- Lin - refers to the absence or low expression of markers associated with lineage committed cells, including but not limited to, T cells (such as CD2, CD3 or CD8); B cells (such as CD10, 19 or 20); myelomonocytic cells (such as CD14, 15, 16); natural killer (“NK”) cells (such as CD2) and red blood cells (“RBC”) (such as glycophorin + ) megakaryocytes, mast cells, eosinophils and basophils.
- T cells such as CD2, CD3 or CD8
- B cells such as CD10, 19 or 20
- myelomonocytic cells such as CD14, 15, 16
- NK natural killer
- RBC red blood cells
- CD34 hi cells Further fractionation of CD34 hi cells to obtain greater enrichment in stem cell activity may be accomplished by any method known in the art.
- Phenotypes that have been reported in the literature to define the most primitive hematopoietic cells include CD34 + /Thy-1 + (Tsukamoto et al., U.S. Pat. No. 5,061,620), CD34 + /HLA-DR lo , CD34 + /CD38 lo , CD34 + /CD45 RA - , and CD34 + /rhodamine 123 lo .
- CD34 hi cells are preferably purified from fetal bone marrow or fetal liver.
- Other fetal, neonatal (particularly cord blood), or adult hematopoietic cell sources including bone marrow, liver, embryonic yolk sac, spleen, and blood may also be used.
- Bone marrow cells may be obtained from the tibia, femur, spine, or other bone cavities.
- CD34 + cells are easily and reproducibly fractionated into CD34 hi and CD34 lo cells based on CD34 antigen density on the cell surface. This is preferably accomplished by fluorescence activated cell sorting (FACS), especially FACS employing a limited panel of antibodies to highly autofluorescent myeloid cells to increase sorting resolution.
- FACS fluorescence activated cell sorting
- Flow Cytometry and Sorting ed. Melamed, Lindmo, and Mendelsohn, Wiley-Liss, Inc., 1990, especially the articles by Lindmo et al., pp. 145-169, and Visser, pp. 669-683.
- a single step selection for CD34 hi cells by flow cytometry will generally achieve an enriched CD34 hi preparation having at least about 0.3% stem cells.
- FACS more preferably multi-color analysis using FACS, is employed to identify and/or select CD34 hi cells present in a cell population.
- the antibody for CD34 may be labeled with one fluorochrome, while antibodies specific for the various dedicated lineages, if used, may be conjugated to a different fluorochrome.
- Fluorochromes which may find use in a multi-color analysis include, but are not limited to, phycobiliproteins, e.g., phycoerythrin and allophycocyanins, fluorescein and Texas red.
- CD34 hi cells may be further fractionated to achieve even more highly purified stem cell populations by subjecting a preparation of CD34 hi cells to additional selection for cell surface markers (or other characteristics) associated with stem cells or against markers associated with lineage committed or mature hematopoietic cells. Additional selections may be performed in separate selection steps or several cell surface markers or may be selected for (or against) in a single step.
- CD34 hi cells are obtained by flow cytometry
- a preliminary separation may be employed to remove lineage committed cells (e.g., T cells, pre-B cells, B cells, and myelomonocytic cells, or minor cell populations, such as megakaryocytes, mast cells, eosinophils and basophils) and enrich the cell population for CD34 hi cells before directly selecting for CD34 hi cells.
- lineage committed cells e.g., T cells, pre-B cells, B cells, and myelomonocytic cells, or minor cell populations, such as megakaryocytes, mast cells, eosinophils and basophils
- platelets and erythrocytes are removed prior to sorting. It is not essential to remove every dedicated cell class, particularly minor cell populations.
- Preliminary separations may conveniently be performed, for example, using magnetic beads coated with one or more specific monoclonal
- Dead cells may be selected against by employing such dyes as propidium iodide.
- Stem cells have low side scatter and low forward scatter profiles as determined by FACS analysis. Cytospin preparations show that stem cells have a size between mature lymphoid cells and mature granulocytes. Cells may be selected based on light-scatter properties as well as their expression of various cell surface antigens.
- Monoclonal antibodies are particularly useful for identifying cell surface markers (membrane proteins exposed on the cell surface and readily identified, e.g., by specific antibodies) associated with particular cell lineages and/or stages of differentiation.
- the antibodies may be attached to a solid support to facilitate preliminary separation. The separation techniques employed should maximize the retention and viability of the fraction to be collected.
- Antibodies employed for cell separations may be labeled by any method known in the art.
- Useful labels include, but are not limited to, fluorochromes, biotin, or other widely used labels.
- antibodies may be affixed to a solid support such as magnetic beads, which allow for direct separation.
- Separations can also be effected by exploiting differences in physical properties (e.g., density gradient centrifugation and counter-flow centrifugal elutriation) and vital staining properties (e.g., rho123 and Hoechst 33342).
- Techniques providing more accurate separation include, but are not limited to, FACS, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
- the bone may be flushed with an appropriate balanced salt solution, preferably supplemented with fetal calf serum (FCS) or other source of proteins, in conjunction with an acceptable buffer at low concentration, generally from about 5-25 mM.
- Convenient buffers include, but are not limited to, Hepes, phosphate buffers, and lactate buffers. Otherwise bone marrow may be aspirated from the bone in accordance with conventional methods.
- the bone marrow cells When antibodies are used for positive or negative selection of CD34 hi cells from bone marrow, the bone marrow cells are typically incubated for a short period of time at reduced temperatures, generally about 4° C., with saturating levels of antibodies specific for CD34 and/or other cell surface markers. The cells are then washed with a salt solution plus proteins and suspended in an appropriate buffered medium, then separated by means which recognize bound antibodies specific for particular cell surface antigens.
- Isolated CD34 hi cells may be propagated in a medium containing maintenance factors supporting the proliferation of stem cells, such as the growth factors secreted by stromal cells, which can be obtained from bone marrow, fetal thymus or fetal liver and which can be allogeneic or xenogeneic. For that reason, isolated CD34 hi cells may be propagated by growth in media conditioned by stromal cells or by coculturing with stromal cells. Stromal cells used in such cocultures may be clonal cell lines, e.g., AC3, AC6, or, preferably SyS-1 (see Baum et al., "Long-Term In Vitro Lymphocyte Cultures," copending patent application U.S. Ser. No.
- hematopoietic cells can be removed by employing appropriate monoclonal antibodies conjugated with toxin, antibody and complement, etc., and then selecting for the ability to maintain human stem cells.
- CD34 hi cells may be frozen in liquid nitrogen and stored for long periods of time in 10% DMSO, 50% FCS, 40% RPMI 1640 medium. Once thawed, the cells may be grown in an appropriate culture system.
- CD34 hi cells are the following.
- CD34 hi cells are useful for identifying culture conditions or biological modifiers such as growth factors which promote or inhibit such biological responses of stem cells as self-regeneration, proliferation, commitment, differentiation, and maturation. In this way one may also identify, for example, receptors for these biological modifiers, agents which interfere with the interaction of a biological modifier and its receptor, and polypeptides, antisense polynucleotides, small molecules, or environmental stimuli affecting gene transcription or translation.
- a CD34 hi cell is plated as a single cell or in bulk culture in an appropriate culture system along with the test sample and allowed to expand to produce progeny cells.
- the proliferation, differentiation, and maturation of the CD34 hi cell(s) is compared to that of a CD34 hi cell(s) cultured under control conditions.
- the capacity of stem cells in a CD34 hi population to differentiate into various hematopoietic lineages may be demonstrated by culturing the cells under appropriate conditions, such as those described in the Examples.
- the cells are typically grown on mouse or human stromal cells.
- the medium employed for the culturing of stem cells for these purposes is preferably a defined enriched medium, such as IMDM (Iscove's Modified Dulbecco's Medium) or a 50:50 mixture of IMDM and RPMI (a commonly used medium whose name refers to "Roswell Park Memorial Institute"), and is generally composed of salts, amino acids, vitamins, 5 ⁇ 10 -5 M 2-mercaptoethanol (2-ME), streptomycin/penicillin at 100 ⁇ g/ml and 100 U/ml, respectively, and 10% FCS.
- the medium is typically changed from time to time, generally at least about once or twice per week.
- the capacity of stem cells in a CD34 hi population to differentiate into myeloid cells may be determined as set forth in the Examples below. Alternatively, Dexter-type cultures (containing hydrocortisone) are used; for production of B lymphocytes, Whitlock-Witte type cultures lacking hydrocortisone are used. The capacity to produce both myeloid cells and B lymphocytes may be demonstrated, for example, by culturing stem cells on an appropriate medium containing hydrocortisone and observing the production of myeloid cells, then transferring the cells to a culture lacking hydrocortisone and observing the production of B cells.
- the stem cell population to be tested is cultured for six weeks in a medium comprising a 50:50 mixture Of RPMI 1640 and IMDM containing 10% FCS, 10% horse serum, streptomycin/penicillin, glutamine and 5 ⁇ 10 -7 M hydrocortisone. In the absence of progenitor cells, all mature cells would be expected to die. If at the end of six weeks myeloid cells are observed, one may conclude that there were one or more progenitor cells in the culture which continuously differentiated into myeloid cells. One may then replace the medium with one lacking hydrocortisone to encourage the growth of B cells.
- the presence of B cells indicates that the progenitor cells which were previously capable of producing myeloid cells are also capable of producing B cells.
- the presence of myeloid cells or B cells may conveniently be determined, for example, by FACS analysis.
- isolated fetal thymus fragments are cultured for 4 to 7 days at about 25° C. in order to substantially deplete the thymus of its lymphoid population.
- Stem cells having human leukocyte antigen (HLA) mismatched with the HLA of the thymus cells are microinjected into the thymus tissue, which is then transplanted into a scid/scid mouse as described in EPA 0 322 240, preferably under the kidney capsule.
- HLA human leukocyte antigen
- the capacity of the stem cells to differentiate into erythroid cells may be determined by conventional techniques to identify burst forming units-erythroid (BFU-E) activity, for example, methylcellulose culture (Metcalf (1977) in Recent Results in Cancer Research 61, Springer-Verlag, Berlin, pp. 1-227).
- the present invention makes it possible to prepare relatively large numbers of hematopoietic stem cells for use in assays for the differentiation of stem cells into various hematopoietic lineages.
- These assays may be readily adapted in order to identify substances such as growth factors which, for example, promote or inhibit stem cell self-regeneration, commitment, or differentiation.
- Identification of target antigens associated with a specific hematopoietic cell type One may also use such cells to identify cell surface antigens or other target antigens present in, and preferably specific for, a given hematopoietic cell type. This may be accomplished, for example, by using the cell as an antigen for the production of monoclonal antibodies, which can be screened to obtain those monoclonal antibodies which are specific for the cell type. Such monoclonal antibodies would themselves be useful, e.g., for improved assays, for selecting for cells expressing their target antigen, or for purifying the target antigen itself.
- Gene cloning strategies One may also use such cells to identify and clone genes whose expression is associated with proliferation, commitment, differentiation, and maturation of stem cells or other hematopoietic cells, e.g., by subtractive hybridization or by expression cloning using monoclonal antibodies specific for target antigens associated with these biological events or characteristic of a hematopoietic cell type.
- hematopoietic cells Reconstituting hematopoietic cells or providing cell populations enriched in desired hematopoietic cell types.
- the availability of CD34 hi cells is also useful for reconstituting the full range of hematopoietic cells in an immunocompromised host following therapies including, but not limited to, radiation treatment or chemotherapy.
- Such therapies destroy hematopoietic cells either intentionally or as a side-effect of bone marrow transplantation or the treatment of lymphomas, leukemias and other neoplastic conditions, e.g., breast cancer.
- CD34 hi cells are useful as a source of cells for specific hematopoietic lineages.
- the maturation, proliferation and differentiation of CD34 hi cells into one or more selected lineages may be effected through culturing the CD34 hi cells with appropriate factors including, but not limited to, erythropoietin (EPO), colony stimulating factors, e.g., GM-CSF, G-CSF, or M-CSF, SCF, interleukins, e.g., IL-1, -2, -3, -4, -5, -6, -7, -8, -13, etc., or with stromal cells or other cells which secrete factors responsible for stem cell regeneration, commitment, and differentiation.
- EPO erythropoietin
- colony stimulating factors e.g., GM-CSF, G-CSF, or M-CSF
- SCF erythropoietin
- interleukins e.g., IL-1, -2,
- CD34 hi cells are also important targets for gene therapy.
- Expression vectors may be introduced into and expressed in autologous or allogeneic CD34 hi cells, or the genome of CD34 hi cells may be modified by homologous or non-homologous recombination by methods known in the art. In this way, one may correct genetic defects in an individual or provide genetic capabilities naturally lacking in stem cells. For example, diseases including, but not limited to, ⁇ -thalassemia, sickle cell anemia, adenosine deaminase deficiency, recombinase deficiency, and recombinase regulatory gene deficiency may be corrected in this fashion.
- Diseases not associated with hematopoietic cells may also be treated, e.g., diseases related to the lack of secreted proteins including, but not limited to hormones, enzymes, and growth factors.
- diseases related to the lack of secreted proteins including, but not limited to hormones, enzymes, and growth factors.
- Inducible expression of a gene of interest under the control of an appropriate regulatory initiation region will allow production (and secretion) of the protein in a fashion similar to that in the cell which normally produces the protein in nature.
- a ribozyme, antisense RNA or protein may express in a CD34 hi cell a ribozyme, antisense RNA or protein to inhibit the expression or activity of a particular gene product.
- Drug resistance genes including, but not limited to, the multiple drug resistance (MDR) gene, may also be introduced into CD34 hi cells, e.g., to enable them to survive drug therapy.
- MDR multiple drug resistance
- the CD43 hi cells can be genetically modified to produce an antisense RNA, ribozyme, or protein which would prevent the proliferation of a pathogen in CD34 hi cells or differentiated cells arising from CD34 hi cells.
- mice monoclonal antibodies were used in these studies and those described below: anti-CD34 (Tuk-3) and FITC-labeled Fab'2 anti-Tuk3 (A. Ziegler, University of Berlin, Germany); FITC- or PE-conjugated anti-CD2 (Leu-5b), anti-CD20 (Leu-16), anti-CD19 (Leu-12), anti-CD-14 (Leu-M3), anti-CD15 (Leu-M1), anti-CD16 (Leu-11a), anti-CD33 (Leu-M9), Anti-CD4 (Leu-3a), anti-CD34 (HPCA-2) (Becton Dickinson, Mountain View, Calif.); FITC- or PE-conjugated anti-glycophorin A (D2.10) (AMAC, Westbrook, Me.); PE-conjugated RT6-CD1a (Coulter, Hialeah, Fla.); Tricolor (TC)-conjugated CD45, TC-CD
- Exp. Med., 149:576 was used); goat anti-mouse IgG1-PE antibody (Caltag, San Francisco, Calif.); FITC-conjugated anti-HLA antibodies MA2.1, BB7.2, GAP-A3, and PE-conjugated W6-32 anti-monomorphic class I MHC molecules were derived from hybridomas obtained at ATCC (Rockville, Md.); irrelevant mouse IgG1 (MOPC21) and irrelevant mouse IgG3 (FLOPC21) (Sigma, St. Louis, Mo.). For CD34 staining, Texas Red (TR)-conjugated polyclonal goat anti-mouse IgG3 (Southern Biotechnology Associates, Birmingham, Ala.) was used.
- Cells were washed twice in SB, then incubated for 20 minutes with TR-conjugated goat anti-mouse IgG3 antibodies and FITC-labeled CD14, CD15, CD16 antibodies (hereafter referred to as "Lin") recognizing lineage-committed cells, followed by three washes in SB.
- Cells were resuspended in SB containing 1 ⁇ g/ml propidium iodide (Molecule Probes, Eugene, Oreg.) and sorted using the FACStar Plus cell sorter (Becton Dickinson, San Jose, Calif.). Live cells (i.e., those excluding propidium iodide) that were Lin - were sorted according levels of CD34 expression.
- Sort gates were set based on the mean fluorescence intensity of the isotype control sample. All cells with CD34 values between 10 and 100 times the mean fluorescence value of the isotype control were sorted as CD34 lo . Those cells with values for CD34 that were greater than 100 times the isotype control values were sorted as CD34 hi . Cells were collected in 24 or 48 well plates in RPMI with 2% FCS and 10 mM HEPES and were counted and reanalyzed for purity in every experiment.
- Phenotypic analysis of low density fetal bone marrow revealed that CD14 + monocytes, CD15 + granulocytes, CD16 + granulocytes, and natural killer (NK) cells together comprise an average of 40% ⁇ 9% of all cells, and that these markers identified all highly autofluorescent cells and those with high orthogonal light scatter. These cells, as well as CD2 + T-cells, CD20 + B-cells, and glycophorin A + erythroblasts were distributed largely (>90%) into the CD34 - compartment of the fetal bone marrow.
- CD14 + , CD15 + , and CD16 + i.e., Lin + ) cells expressed antigens associated with mature lineages and had no long term in vitro or in vivo hematopoietic activity.
- Lin + cells were excluded from our FACS analysis by electronic gating without compromising the integrity of the CD34 + compartment.
- FIG. 1 shows the distribution of CD34 and lineage markers as determined by flow cytometry on an (A) isotype control and (B) on low density fetal bone marrow cells stained 20 with anti-CD34 antibody (Tuk-3) and lineage antibodies (CD14, CD15, CD16).
- FIG. 1(B) shows that there are two populations of CD34 + cells, those expressing high levels of CD34 (arrow) and those expressing relatively low levels of CD34 (box).
- CD34 lo An average of 80% of the CD34 + /Lin - cells stained with a mean fluorescence value (MFV) 10- to 100-fold greater than that of the isotype control and are herein referred to as "CD34 lo ".
- CD34 hi The remaining CD34 + /Lin - cells stained with a MFV greater than 100-fold above the control, and are herein referred to as "CD34 hi ".
- Table 1 shows the percentage of CD34 + , CD34 - , CD34 hi , and CD34 lo subpopulations of low density, Lin - cells from 15 individual fetal bone marrow isolates.
- the percent of CD34 + cells was determined by measuring all cells that stain above 99% of the isotype control (the remainder being CD34 - ).
- the percentage of cells that are CD34 hi and CD34 lo was determined by the relative density of the CD34 antigen on the cell surface relative to the isotype control. While the percentage of low density Lin - fetal bone marrow cells that occupy the CD34 hi and CD34 lo compartments varies (4.6% ⁇ 3.5 and 21% ⁇ 6.6, respectively), the average staining intensities are highly reproducible.
- Table 2 shows the mean fluorescence intensity for CD34 antigen staining for nine fetal bone marrow samples as analyzed by flow cytometry, comparing the value of CD34 hi and CD34 lo populations to that of the isotype controls.
- the log of the ratio of the mean fluorescence intensity compared to the isotype control value is consistently greater than 2 for CD34 hi and between 1 and 2 for CD34 lo cells.
- Identical findings have been obtained with direct staining using a different anti-CD34 antibody (HPCA-2).
- FIG. 2 shows a histogram of the CD34 fluorescence distribution of CD34 hi and CD34 lo sorted by flow cytometry. Sorted CD34 lo and CD34 hi cells were reanalyzed and the percentage overlap calculated by the Overton subtraction procedure using Multiplus Flow Cytometric Histogram software (Phoenix Flow Systems, San Diego, Calif.). The shaded portion of the curves indicates the non-overlapping portion of the two populations and hence the relative purity of the samples. These measurements consistently gave an estimate of 85% to 95% purity; therefore 5 to 15% contamination of CD34 lo with CD34 hi , and vice versa, was expected. Such measurements proved important for quantitative studies on populations with extremely high proliferative potential, as described below.
- Bone marrow samples were stained and sorted as described above. Twenty to fifty thousand sorted cells were stained with a panel of PE- or FITC-conjugated monoclonal antibodies as described, then analyzed on a FACScan fluorescent cell analyzer (Becton Dickinson). A portion of each sorted population was incubated with the appropriate isotype control to establish the background level. The percent positive cells was determined relative to the isotype control by subtracting the background value from the experimental value.
- CD34 hi and CD34 lo subsets were characterized by their differential expression of a limited panel of lineage specific antigens or antigens that have been used by other groups to describe stem cells (FIG. 3).
- CD34 hi cells express low levels of CD13 and CD33, are enriched for cells expressing low to intermediate levels of HLA-DR, CD38 and CD45RA, and have no detectable CD2, 10, or 19.
- CD34 lo cells express high amounts of CD38 and HLA-DR, low levels of CD19 and CD10, and no detectable CD33 or CD13.
- CD34 hi and CD34 lo subsets are distinct cell populations, and that CD34 hi cells are enriched in primitive hematopoietic cells as judged by expression of various cell surface antigens.
- CD34 lo population appears more mature and contains a large percentage of CD10 + CD19 + pre-B-cells.
- the relative stem cell content of each population was determined by limiting dilution analysis and/or single cell plating of cells seeded onto pre-established murine stromal cell monolayers in 96 well plates as previously described (Baum et al. (1992) Proc. Natl. Acad. Sci. USA 89:2804-2808.
- FIG. 4 lists the precursor frequencies for fetal bone marrow subpopulations as percent responding cells in SyS-1 coculture. Limiting dilution analysis of sorted fetal bone marrow was employed to establish the frequency of cells that form cobblestone areas between weeks four and six of culture. Some cultures were either lost to contamination or sacrificed for analysis prior to week 6 (blank spaces). The average response (AVG) and standard deviation (SD) were calculated. The number of cells containing one cobblestone area-forming cell is listed as the reciprocal of the average frequency. (1/AVG). Significant differences were observed in the growth kinetics of individual tissues, but the growth rate of all populations appeared to decline after six weeks of culture.
- the CD34 + population gave an average frequency of 1/646.
- the CD34 hi cell population had a 3.4- to 4-fold increase in precursor frequency over the CD34 + population, which approaches a quantitative recovery of all of the activity in the CD34 hi compartment. It was also confirmed that the CD34 - and Lin + populations had no activity in this assay.
- FIG. 5 shows phenotypic analyses of bulk cultures of CD34 hi cells.
- CD34 hi cells were cultured on a stromal cell layer for six weeks, then the entire culture was harvested and stained with either the appropriate isotype matched antibodies (A), antibodies to the B cell progenitor surface markers CD19 and CD10 (B), or antibodies to myeloid progenitor cell markers CD15 and CD33 (C).
- Stromal cells and dead cells were excluded by light scatter and dye exclusion gating. As shown in FIG. 5, approximately 80% of the wells analyzed showed populations expressing both B and myeloid cell markers.
- SCID-hu bone assay female C.B-17 scid/scid (SCID) mice were bred under sterile conditions and protected by antibiotic treatment in drinking water (sulfamethoxazole and trimethoprim, 400 and 80 mg/kg mouse/wk, respectively). Mice between 6 to 8 weeks of age were used. Human fetal long bones obtained as mentioned above were split lengthwise and transversely cut in half to yield 4 bone fragments per long bone. These fragments were immediately implanted subcutaneously into the SCID mice mammary fat pads. Usually two bone pieces are engrafted into each mouse.
- SCID-hu bone mice were irradiated with 350 rads in a single dose dispensed with a 1500 Ci 137 Cs source using a 30% attenuation shielding (J. L. Sheperd & Assoc., San Francisco, Calif.). Experiments were performed to determine that SCID mice could tolerate a dose of total body irradiation up to 400 rads, considerably below the level that normal healthy mice can tolerate. At doses of 350 to 400 rads, engraftment of donor-derived cells reached a level of greater than 50%, usually 80%. Sorted cells were then injected directly into the bone using a Hamilton syringe in a 10 ⁇ l volume.
- SCID-hu bone mice were kept for 5 to 9 weeks, then sacrificed by cervical dislocation. Human bones were removed and adherent tissues dissected away. The bones were split open in order to flush the marrow cavity with SB. Collected cells were spun down and the pellet was resuspended for 10 minutes into a red blood cell lysing solution (Kyoizumi et al. (1992) to lyse red blood cells. Cells were washed twice and counted before being stained by two-color immunofluorescence with directly labeled antibodies against HLA in combination with anti-CD19, -CD20, -CD33, -CD14, and -CD34. Grafts with low numbers of cells may be pooled to facilitate staining. FITC- and PE-conjugated irrelevant mouse immunoglobulins were used as negative controls. Analysis was performed on a FacScan fluorescence activated cell scanner (Becton Dickinson).
- SCID mice After the SCID mice had been implanted with human bone fragments, they were allowed to recover for a minimum of 5 weeks. They were then subjected to total body irradiation to deplete the implanted bones of hematopoietic cells. Immediately following irradiation, 1.5 to 5 ⁇ 10 4 sorted fetal CD34 hi and CD34 lo cells were directly injected into the bone cavity. Cells were sorted against an extended Lin panel which in addition to CD14, CD15, and CD16 included CD2, CD20, and glycophorin in order to ensure complete depletion of mature committed cells or cells with a potentially detrimental effect in such an allogeneic setting.
- Table 3 presents the results of the reconstitution of SCID-hu bones by CD3.4 sets in three distinct experiments in which the human bones were injected with 1.5 to 5 ⁇ 10 4 fetal bone marrow CD34 subsets or noninjected. Grafts were retrieved 5 to 9 weeks after injection, cells counted and stained for the presence of CD45 + /HLA class I + human and donor cells. The cell numbers retrieved from grafts varied, but there were no significant differences in the overall cellularity (i.e., cell number) of the bones whether they were injected with CD34 hi /Lin - or CD34 lo /Lin - fetal bone marrow cells or uninjected.
- FIG. 6 shows the results from staining a representative SCID-Hu bone graft reconstituted with allogeneic CD34 hi fetal bone marrow cells and retrieved after nine weeks. 96% of the cells coexpressed CD45 and W6-32, indicating they were human cells. 91% of the cells coexpressed the anti-polymorphic HLA of the donor in combination with W6-32, indicating that they were donor derived (top left panels). Gating on the donor population revealed a heterogeneous distribution of bone marrow cells, typically indicative of the presence of multiple lineages (upper right panel). B-cells were clearly identifiable by expression of CD19 and CD20.
- Donor derived CD19 + represented 57% of the total sample and CD20 + represented 42%. Gating on CD20 + donor cells showed forward (FSC) and side scatter (SSC) characteristics of B-cells (middle right panel). CD33 + donor cells were also found and were observed to distribute in the expected myeloid region of bone marrow cells (bottom left panel). Also, CD14 + monocytes could be identified with characteristic size and granularity features (lower middle right panel). Donor-derived CD33 + represented 30% of total sample and CD14 + represented 11%. A population of CD34 + donor cells was identifiable (14% of total sample); the specificity of staining was controlled by lack of immunoreactivity with irrelevant antibodies (bottom right panel).
- CD34 lo cells never engrafted, so no donor progeny could be identified and the phenotypic profile of the grafts was identical to that of noninjected controls, showing only the recovery of host hematopoiesis.
- Fetal bone marrow CD34 - cells were tested and likewise showed no engraftment. Because of the initial sort purities and because donor-derived myeloid cells were retrieved after nine weeks, these results argue against maintenance or expansion of mature cells but strongly demonstrates multilineage differentiation from the CD34 hi stem cell-containing population.
- Human fetal CD34 + cells can reconstitute a depleted allogeneic thymus cultured in vitro or implanted into SCID mice and generate donor-derived thymocytes (Galy et al. (1993) J. Exp. Med. 178:391-401; Peault et al. (1991) J. Exp. Med. 176:1283-1286).
- the SCID mouse model allows maintenance of donor-derived T-cells for as long as 4.5 months.
- a three-color immunostaining procedure was used to stain thymocytes recovered from thymic grafts to assess the quality of donor-derived thymopoiesis by examining the coordinated expression of CD1a, CD3, CD4 and CD8 molecules.
- Thoroughly depleted thymic grafts were reconstituted with CD34 subsets from allogeneic fetal bone marrow and analyzed after 6 to 14.5 weeks.
- fragments were irradiated with 250 rads given without attenuation in a single dose on a 137 Cs source irradiator (J. L. Shepherd & Assoc.). Fragments were washed and immediately microinjected with the HLA mismatched sorted cell populations in a 1 ⁇ l volume using an oil-filled microinjector (Narishige, Japan) and 1 mm diameter glass micropipets (World Precision Instruments, Sarasota, Fla.). Fragments were placed back on the filters and incubated at 37° C. with 5% CO 2 overnight, then inserted under the kidney capsule of anesthetized 6 to 8 week-old SCID mice.
- mice were sacrificed by cervical dislocation at various times after the transplantation, and the thymus grafts were recovered, reduced to a cellular suspension, and subjected to a three-color immunofluorescence analysis on the FACScan, using mAbs directly labelled with FITC, PE and TR. Grafts with low numbers of cells may be pooled to facilitate staining. Samples were analyzed on the FACScan to determine the proportion of human and donor-derived cells (combination of HLA of donor, anti-class I monomorphic and CD45) and the quality of the thymopoiesis (combination of HLA of donor and CD1a plus CD3, or CD4 plus CD8).
- FIG. 8 Further phenotypic analysis (FIG. 8) showed that the T-cell progeny of CD34 hi fetal bone marrow cells closely resemble those of normal fetal thymocytes, based on the high expression of CD1a, graded levels of CD3 staining, and co-expression of CD4 and CD8 on the majority of thymocytes, although there were a small number of single positive CD4 or CD8 cells.
- the graft injected with CD34 hi cells showed complete reconstitution with thymocytes coexpressing HLA class I monomorphic and specific determinants of the donor.
- the graft injected with the CD34 lo subset was not reconstituted with any donor cells, and all thymocytes recovered were of host origin, having matured to express very high amounts of class I antigens and of CD3, no CD1; there were no cells positive for both CD4 and CD8.
- only one of nine recovered grafts injected with CD34 lo cells contained donor cells.
- this graft contained only donor-derived mature thymocytes. After 12 weeks, host thymocytes had completely differentiated into MHC class I bright cells with high levels of CD3 without CD1 or co-expression of CD4 and CD8.
- CD34 hi fetal bone marrow cells were capable of engrafting an allogeneic thymus and generating T-cells for sustained periods of time. It was also confirmed that CD34 - fetal bone marrow cells were devoid of pre-T-cell activity. Taken together, these data clearly show that the capacity to generate T, B, and myeloid cells is exclusively restricted to the CD34 hi compartment of the fetal bone marrow.
- hematopoietic progenitor cell or stem cell
- the hallmark of a very early hematopoietic progenitor cell (or stem cell) is the ability to differentiate into multipotent progenitors and generate long term hematopoiesis in immunocompromised hosts.
- Cocultivation of primitive progenitor cell populations on marrow-derived stromal cells has been shown to maintain active hematopoiesis for extended periods of time (8-12 weeks).
- Long term stromal coculture assays have been extensively used to determine the hematopoietic stem cell content of candidate populations.
- the myelo-erythroid potential is generally assayed and direct evidence is often lacking to correlate this activity with primitiveness, particularly in the lymphoid lineage.
- the present disclosure demonstrates the in vitro generation of CD19 + B cells from most tissues tested for up to 8 weeks of culture. Myelopoiesis was evident in the same cultures with expression of CD33. Cells found after 6 weeks of culture likely arose from primitive hematopoietic stem cells for a number of reasons. First, there was no detectable CD10 or CD19 on the surface of CD34 hi cells, ruling out the possibility that early B cell progenitors contaminating the starting population had been maintained. Second, the culture conditions used did not support the maintenance of the CD10 or CD19 positive CD34 lo population over the 6 weeks of culture; thus the expansion of contaminating lineage-committed progenitors is unlikely.
- thymic piece implanted alone engrafts very poorly and does not generate immature thymocytes (CD1 + ) past 6 weeks, unless a source of progenitors is added in the form of a fetal liver fragment or microinjected stem cells.
- thymus itself does not seem to contain stem cells or rapidly exhausts the small number of stem cells that it may contain.
- CD34 hi cells on SyS-1 stroma revealed that the stem cell activity was contained in about 0.5-1% of CD34 hi cells, which represents a 100-fold increase over whole bone marrow and a 3-5-fold increase over CD34 + cells.
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Also Published As
Publication number | Publication date |
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CA2170357A1 (fr) | 1995-03-02 |
AU685506B2 (en) | 1998-01-22 |
AU7676994A (en) | 1995-03-21 |
EP0722331A4 (fr) | 1997-10-01 |
WO1995005843A1 (fr) | 1995-03-02 |
EP0722331A1 (fr) | 1996-07-24 |
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